Research and diagnostic procedures benefit from rapid, accurate, and qualitative and/or quantitative determinations of substances (“analytes”) that are present in biological samples, such as biological tissues or fluids, at low concentrations. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, prostaglandins or infectious organisms in blood, urine, saliva, dental plaque, gingival crevicular fluid, or other biological specimens is desirably determined in an accurate and rapid fashion for suitable diagnosis or treatment.
In many cases, an analyte is identified in a sample using a compound that specifically recognizes the chemical features of the analyte. Often, monoclonal antibodies specific for one or more chemical epitopes on an analyte are used. In other cases, oligonucleotides that are specific for gene transcripts (e.g., mRNA analytes) can be used to assess gene expression in a cell-containing sample.
The complex formed between the antibody (or oligonucleotide) and analyte can be detected by a variety of known methods. The most commonly used methods employ a signal generating moiety of some type which is either already attached to the antibody/oligonucleotide, or becomes attached to the antibody/oligonucleotide through further reaction. For example, in the formation of a complex of biotin with avidin, the complex may be detected using a label on either the avidin or biotin molecule. Such a label may be a radioisotope or an enzyme conjugated to the avidin or biotin. Alternatively, the avidin-biotin complex might be detected by further reaction with a labeled molecule which is specific to either or both parts of the complex. It is commonly known to do the same with antigens and their corresponding antibodies.
During an analysis procedure, the specific binding ligand of interest (such as an antigen from an infectious agent) is often detected using colorimetric, fluorescent, or chemiluminescent signals resulting from reaction of the enzyme label with its corresponding substrate.
In many colorimetric assays, a sample containing analyte is mixed with detection reagents and a colored product is produced if the analyte is present. Analysis can be carried out for a predetermined time after the assay is started (end point analysis), or can be performed over time to assess the generation of color (kinetic analysis). Both types of assays are directed at determining the amount of analyte in the sample. For endpoint analysis, the intensity of the colored reaction (e.g., as measured by the absorbance value of the colorimetric reaction product) is directly proportional to the amount of analyte present. Here, the presence of a large amount of analyte can result in a more intense color. For kinetic analysis, the time required before the appearance of a color (reaction time) is inversely proportional to the amount of analyte present. Here, the presence of a large amount of analyte can result in a rapid reaction. The concentration of analyte in test samples can be calculated from a standard curve.
Some problems associated with colorimetric analysis are that colorimetric reagents can have chemical properties causing them to exhibit poor solubility in assay compositions. Excipient components intended to improve solubility characteristics can potentially interfere with one or more aspects of the assay. Such interference can yield results that do not accurately reflect the amount of analyte in the sample or can significantly slow the reaction time.